Chloride channel function in the yeast TRK-potassium transporters
Chloride channel function in the yeast TRK-potassium transporters
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DOI：
10.1007/s00232-004-0671-1
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发表时间：
2004-04-01
影响因子：
2.4
通讯作者：
Rivetta, A
中科院分区：
文献类型：
--
作者：
Kuroda, T;Bihler, H;Rivetta, A
Trk蛋白Trk1p和Trk2p是酿酒酵母主动积累钾的主要因子。在以前的研究中，通过全细胞膜片钳测量的通过这些蛋白质的内向电流被证明对细胞外钾浓度的变化非常不敏感，尽管它们确实随着细胞外质子浓度的增加而增加--正如预期的那样，H+偶联到K+吸收。这些令人费解的观察结果现已得到更详细的探索，主要发现如下：a)大的内向TRK电流不是由K+或H+内流携带的，而是由氯离子外流携带的；b)在钾充足的细胞中，Trk1p和Trk2p的正常表达水平下，内向TRK电流大约一半由Trk1p贡献，一半由Trk2p贡献；但c)应变背景强烈影响这些电流的绝对大小，在W303来源的球体中几乎是S288c来源细胞的两倍(相同的细胞大小和相同的记录条件)；D)加入增加细胞大小的突变(高尔基钙泵Pmr1p的缺失)或上调TRK2启动子的突变，可进一步显著增加TRK电流；e)去除细胞内氯(例如，被硫酸盐或葡萄糖酸盐取代)显示出小的内向电流，该电流依赖于K+，并可因K+饥饿而增强；和f)最后，后者表现出两个饱和动力学分量，初步估计在46 PM[K+](OUT)和6.8 mm[K+](OUT)时K-0.5，饱和通量类似于5 mm/min和类似于10 mm/min(指细胞内水)。这些数字分别与Trk1p和Trk2p的正常K+转运特性兼容。
The TRK proteins-Trk1p and Trk2p- are the main agents responsible for "active" accumulation of potassium by the yeast Saccharomyces cerevisiae. In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration-qualitatively as expected for H+ coupling to K+ uptake. These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K+ or H+, but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K+-dependent and can be enhanced by K+ starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K-0.5 at 46 pm [K+](out) and 6.8 mm [K+](out), and saturating fluxes of similar to5 mm/min and similar to10 mm/min (referred to intracellular water). These numbers are compatible with the normal K+-transport properties of Trk1p and Trk2p, respectively.